Vector information sheet.

Vector Name Source Sequence accession/link Description

pNIC28-Bsa4
Opher Gileadi (SGC) pET expression vector with His6 tag in 22-aa N-terminal fusion peptide, with TEV protease cleavage site. Includes sites for LIC cloning, and a stuffer fragment that includes the SacB gene, allowing negative selection on 5% sucrose Kanamycin, 50 g/ml T7 - lacO LIC. (vector treated with BsaI, then with T4 DNA polymerase in presence of dGTP) Supplied in PCR primer MHHHHHHSSGVDLGTENLYFQ*SM (* - TEV cleavage site) 2684.1 Da including Met (2465.8 Da removed by TEV cleavage) supplied in PCR primer TEV DE3 hosts: BL21, Rosetta, etc. MUST express T7 RNA polymerase. pLIC-for: TGTGAGCGGATAACAATTCC pLIC-rev: AGCAGCCAACTCAGCTTCC

Antibiotic resistance Promoter Cloning Initiation codon N-terminal fusion seq. N-terminal fusion MW Termination codons Protease cleavage Additional features Preferred host 5 sequencing primer 3 sequencing primer

T7 promoter
Nde I (41)

lac operator

Lic 5 (96)
Bsa I (106)

Nco I (107)

lac I
SacB

pNIC-28-BsaI
7284 bp

Bsa I (2037)

Lic 3 (2051)

Bam HI (2053)
Not I (2085)
Xho I (2093)

T7 terminator

ColE1 pBR322 origin

f1 origin

kan sequence

Opher Gileadi, Structural Genomics Consortium opher.gileadi@sgc.ox.ac.uk

Polylinker region
pLIC-forward ------------------lac operator ~~~~~~~~~~~~~~~~~~~~~~~~~~~ CTCGATCCCG CGAAATTAAT ACGACTCACT ATAGGGGAAT TGTGAGCGGA TAACAATTCC GAGCTAGGGC GCTTTAATTA TGCTGAGTGA TATCCCCTTA ACACTCGCCT ATTGTTAAGG NdeI ~~~~~~ M H H H H H CCTCTAGAAA TAATTTTGTT TAACTTTAAG AAGGAGATAT ACATATGCAC CATCATCATC GGAGATCTTT ATTAAAACAA ATTGAAATTC TTCCTCTATA TGTATACGTG GTAGTAGTAG Upper-LIC BsaI ~~~~~~~~~~~~~~~~ ______ H S S G V D L G T E N L Y F Q S ATCATTCTTC TGGTGTAGAT CTGGGTACCG AGAACCTGTA CTTCCAATCC ATGGAGACCG TAGTAAGAAG ACCACATCTA GACCCATGGC TCTTGGACAT GAAGGTTAGG TACCTCTGGC T7-forward -------------------

5101

5161

5221

5281

ACGTCCACAT TGCAGGTGTA

(SacB fragment)

7261

EcoRI BsaI Lower-LIC BamHI ~~~~~~~ SacI ______ ~~~~~~~~~~~~~~~~ ~~~~~~ ~~~~~~ GATATCCTAT TGGCATTGAC GGTCTCCAGT AAAGGTGGAT ACGGATCCGA ATTCGAGCTC CTATAGGATA ACCGTAACTG CCAGAGGTCA TTTCCACCTA TGCCTAGGCT TAAGCTCGAG SalI HindIII ******~~~~~~~ CGTCGACAAG CTTGCGGCCG CACTCGAGCA CCACCACCAC CACCACTGAG ATCCGGCTGC GCAGCTGTTC GAACGCCGGC GTGAGCTCGT GGTGGTGGTG GTGGTGACTC TAGGCCGACG T7-reverse ----------------TAACAAAGCC CGAAAGGAAG CTGAGTTGGC TGCTGCCACC GCTGAGCAAT AACTAGCATA ATTGTTTCGG GCTTTCCTTC GACTCAACCG ACGACGGTGG CGACTCGTTA TTGATCGTAT ------------------pLIC-rev

7321

7381

Primers for LIC cloning: Upstream: add TACTTCCAATCCATG to the 5 end (ATG in-frame with the desired coding sequence). Downstream: add TATCCACCTTTACTG to 5 end of downstream primer; add termination codon, if necessary.

Opher Gileadi, Structural Genomics Consortium opher.gileadi@sgc.ox.ac.uk